Photodamage and the importance of photoprotection in biomolecular-powered device applications.

ABSTRACT

In recent years, an enhanced understanding of the mechanisms underlying photobleaching and photoblinking of fluorescent dyes has led to improved photoprotection strategies, such as reducing and oxidizing systems (ROXS) that reduce blinking and oxygen scavenging systems to reduce bleaching. Excitation of fluorescent dyes can also result in damage to catalytic proteins (e.g., biomolecular motors), affecting the performance of integrated devices. Here, we characterized the motility of microtubules driven by kinesin motor proteins using various photoprotection strategies, including a microfluidic deoxygenation device. Impaired motility of microtubules was observed at high excitation intensities in the absence of photoprotection as well as in the presence of an enzymatic oxygen scavenging system. In contrast, using a polydimethylsiloxane (PDMS) microfluidic deoxygenation device and ROXS, not only were the fluorophores slower to bleach but also moving the velocity and fraction of microtubules over time remained unaffected even at high excitation intensities. Further, we demonstrate the importance of photoprotection by examining the effect of photodamage on the behavior of a switchable mutant of kinesin. Overall, these results demonstrate that improved photoprotection strategies may have a profound impact on functional fluorescently labeled biomolecules in integrated devices.